The sexual cycle of Toxoplasma gondii is limited to the feline intestine where millions of oocysts are formed and subsequently excreted in their feces. The goal of this application is to recapitulate the T. gondii sexual cycle in tissue culture Towards this goal, we have already developed a protocol for harvesting and culturing of polarized feline intestinal epithelial cells. Our goal for this R03 is to immortalize these cat intestinal epithelial cells while maintaining their ability to polarize. We have also already engineered T. gondii parasites to express a negative selectable marker from an asexual stage-specific promoter. For this R03 we will culture these genetically manipulated parasites in the feline intestinal epithetical cells using the negative selection to inhibit the growth of the asexul stages. This inhibition will prevent the rapidly replicating form, called tachyzoites, from lysing he intestinal cells. Inhibition of host cell lysis will give the sexual stages time to develop. Curretly, only a limited number of sexual crosses of T. gondii are performed because they must occur in live cats after feeding them infected mouse brains and collecting their feces 3-10 days after infection. Creation of ex vivo sexual development conditions will allow classical genetic crosses to become routine in laboratories, which will open multiple new experimental design strategies. It will also allow for a molecular analysis of the complete lifecycle of a protozoan parasite, as T gondii asexual development can already be performed in tissue culture. Another benefit of the determining conditions for T. gondii sexual development in tissue culture will be the production of a stable vaccine. A T. gondii vaccine produced as an oocyst will be ideal because it will be stable in any environmental conditions (no refrigeration) and it will be an oral inoculation (no needles). We have been highly successful generating the tools necessary to begin sexual development studies of T. gondii in tissue culture. We will now use these tools to select parasites undergoing sexual development in ex vivo feline intestinal cells. Any progression through the sexual cycle of T. gondii in tissue culture will be a major advance for the field because a molecular analysis of these recalcitrant stages can begin.